The murine 21-hydroxylase (C-21) gene is expressed primarily in the adrenal gland. We propose to further define the features of this gene that direct this tissue-specific expression. Most importantly, however, we propose to identify the regulatory proteins that are expressed in the adrenals that interact with this gene and direct its expression. Specifically, we propose to: 1) Identify the defect in the Y1 cell that prevents production of stable 21-hydroxylase mRNA. 2) Use linker scanning mutagenesis to define the different regions required for 21-hydroxylase-CAT expression in Y1 cells. 3) To test the function of cis-acting regulatory sequences in transgenic mice. 4) Identify derivatives of the Y1 cell line that bear mutations in transacting factor genes. 5) Determine whether transacting factors bind directly to the 21-hydroxylase gene. 6) Clone the genes encoding the transacting factors. Characterization of these proteins and the genes that encode them should provide considerable insight into the process of tissue-specific gene expression.